Preparation of high purity xanthine oxidase from bovine milk

ABSTRACT

Bovine milk xanthine oxidase, referred to herein as XO, is isolated and purified from raw whole milk by a streamline method without the use of proteolytic and lipolytic enzymes, butanol, or other organic solvents. Sodium salicylate, ethylenediaminetetraacetate (EDTA), and a 0.2 M phosphate buffer are added to fresh milk. After incubation at 40°-45° C. for 105 min., the mixture is adjusted to 1-2% with Triton X-100 and allowed to incubate for 15 min. The mixture is cooled to 4° C., followed by a 2-step fractionation of the proteins with ammonium sulfate. The crude enzyme is isolated as a red-brown precipitate which is dissolved in 0.1 M Tris/CaCL 2  buffer and stored for from 12 to 168 hours at -20° C. The isolated enzyme is purified by column chromatography (Sephadex G-75, Sephacryl S-200, Sepharose 6B, and Sephadex G-75). The final stage of purification is accomplished by passing the enzyme preparation through a DEAE-Sephadex A-50 column in a continuous linear salt gradient from 0.005 M to 0.1 M pyrophosphate buffer. 
     The purified enzyme is not denatured and has, on the average, a constant E 280  /E 450  ratio of 4.1, and one symmetric peak by gel chromatography. Analysis by polyacrylamide disc gel electrophoresis demonstrates a single band, while commercially available XO shows 7-14 bands, only one of which contains XO activity (the rest being impurities). The average yield of the final product is about 21% which is 110% higher than the yield of less pure XO obtained using the best prior art.

FIELD OF THE INVENTION

This invention relates to an improved method for producing xanthineoxidase (referred to herein as XO) of high purity from bovine milk.Highly purified XO is presently unavailable commercially, and is neededin research and in a variety of industrial, clinical, and pharmaceuticalapplications.

PRIOR ART

Bovine milk xanthine oxidase (xanthine: oxygen oxidoredactase; E.C.1.2.3.2.) is a conjugated iron-sulfur molybdenum flavoprotein widelydistributed in animals, plants, and microorganisms where it has a rolein purine catabolism. In many animals, including primates, XO is foundin the liver, kidney, blood, intestinal mucosa and milk (Li and Vallee,In Modern Nutrition in Health and Disease: Dietotherapy, 5th Ed. Lea andFebiger, Philadelphia, Pa., pp. 372-399, 1973; Zikakis et al., J. FoodSci. 41:1408-1412, 1976). It catalyzes uric acid production fromhypoxanthine and xanthine in terminal purine catabolism.

Most of the XO in cow's milk is closely associated with the milk fatglobule membrane (MFGM) (Morton, Biochem. J. 57:231-237, 1954). The MFGMhas a protenaceous surface that interfaces with the milk plasma phase onthe exterior and the globule lipids on the interior (Brunner, InFundamentals of Dairy Chemistry, 2nd Ed. AVI Publishing Co., Westport,Conn., pp. 474-602, 1974).

Ball in 1939 (J. Biol. Chem. 128:51-67) was the first to isolate andpartially purify XO from cow's milk by treating buttermilk withpancreatin. From 1955 to the recent past, numerous purification methodsfor cow's milk XO have been described. Pancreatin has been used in XOpurification to degrade casein micelles to lower molecular weightcomponents so that they may be eluted behind XO in subsequentchromatographic fractionations. However, pancreatin is not specific forcasein degradation, and has a proteolytic effect on all proteins (Nelsonand Handler, J. Biol. Chem. 243:5368-73, 1968). Avis et al., (J. Chem.Soc. (London) pp. 1100-1105, 1955) managed to crystallize XO in thepresence of ethanol giving a protein/flavin ratio (a most importantpurity index for XO) of 5.0-5.2. However, the yield was poor and thespecific activity of the product varied widely.

In 1964, Gilbert and Bergel (Biochem. J. 90:350-353) published a methodwhich improved the yield but the product was less pure (E₂₈₀ /E₄₅₀ranged from 5.4-6.0) than that of Avis et al., 1955. Their methodincluded pancreatin digestion of the buttermilk, treatment with butanol,and the addition of EDTA and sodium salicylate. In 1968 Nelson andHandler prepared milk XO by a non-proteolytic method (i.e., withoutpancreatin digestion). They concluded that their preparation washomogeneous, with a E₂₈₀ /E₄₅₀ ratio of 5.1-5.3. Hart et al., (Biochem.J. 116:851-863, 1970) and Nelson and Handler (1968) have indicated thatpurified XO differs according to the purification method, and thatproteolysis adversely affects the enzyme. Waud et al. (Arch. Biochem.Biophys. 169:695-701, 1975), and Nagler and Vartanyan (Biochem. Biophys.Acta 427:78-90, 1976) demonstrated that purification proceduresemploying pancreatin yield XO and sub-units with lower molecular weight,and that the XO migrates faster on polyacrylamide gel electrophoresisthan XO prepared by a non-proteolytic treatment. Furthermore, Nathansand Hade (Biochem. Biophys. Res. Comm. 66:108-114, 1975) showed that XOisolated in the presence of pancreatin copurifies with proteases frompancreatin. The non-proteolytic procedure of Waud et al., (1975) whichcomprises butanol extraction, ammonium sulfate precipitation, andchromatography is the best available method. The final product never hasa E₂₈₀ /E₄₅₀ ratio better than 4.8 and the yield is 10%.

In summary, prior art teaches an incubation of milk buffered with SodiumSalicylate and EDTA; digestion with pancreatin, or extraction withbutanol, washing with an ionic detergent; precipitation of casein withammonium sulfate; further removal of casein by treatment with ammoniumsulfate, and finally purifying on one or two chromatographic columns.

The objective of this invention is to obtain a purer XO than produced byprior art by operating under conditions that improve the final product.The invention features the use of a mild non-ionic detergent,maintaining at low temperature to remove more casein, and the use ofultrafiltration and multiple chromographic columns to concentrate the XOand remove non-XO proteins with lower or higher molecular weights.

SUMMARY OF THE INVENTION

This invention consists of an improved method for the isolation andpurification of XO from bovine raw milk. To assure good yield, thestarting milk is assayed for XO activity before using it. As a rule,good yield is expected when the activity of starting fresh raw milk(which is maintained immediately after milking at 38° C. and assayedwithin 60 min.) is above 60 μl 0₂ /ml/hr. or above 140 μl 0₂ /ml/hr. forraw milk kept at 4° C. for 1-12 hrs. after milking. Sodium salicylateand EDTA are added as enzyme protectors. The mixture is diluted 1:1 withpotassium phosphate buffer and incubated at 40° to 45° C. for 2 hourswith continuous mixing. After 105 min. of incubation, 1% by volume ofTriton X-100 or a similar non-ionic detergent is added and the mixtureis allowed to incubate for 15 min. The use of a non-ionic detergentfacilitates the separation of the XO from MFGM without adding ionicsalts or degrading the enzyme.

The mixture is cooled to 4° C. and all subsequent steps are carried atthat temperature. Solid ammonium sulfate is added (200 gm/liter) to themixture, which is stirred for 15 min., and then centrifuged at 12,225 gfor 20 min. To the resulting yellow supernatant liquid, which containedthe enzyme, solid ammonium sulfate is added (70 gm/liter), stirred for15 min., and centrifuged at 12,225 g for 20 min. The red-brownpercipitate is dissolved in a minimal volume of 0.1 M Tris*/CaCL₂ buffer(pH 7.0) and stored at freezing or below, e.g. -20° C. for from 0.5 to7.0 days. Prior art does not call for storage at that temperature, butthis step precipitates caseins which are the commonest non-XO proteinsin milk.

The frozen preparation is thawed and centrifuged at 12,225 g for 20 min.The active enzyme retained in the supernatant liquid is concentrated ona XM50 microfilter and applied on a Sephadex G-75 column. The elutedfractions are analyzed spectrophotemetrically at 280 and 450 nm andcatalytic activity is measured at 295 nm. Fractions showing activity arepooled, concentrated, and reanalyzed as above. The concentrated sampleis applied on a Sephacryl S-200 superfine or on a Sephadex G-200 column.The eluted fractions are analyzed for absorption and activity, activefractions pooled, concentrated as above, and passed through a Sepharose6B type column. All eluted fractions are tested as above; fractions withactivity are pooled, concentrated as above, and desalted by passagethrough a Sephadex G-75 column. Finally, the active fractions from thiscolumn are pooled, concentrated, and applied on either a DEAE SephadexA-50 or on DEAE Sepharose CL-6B anionic exchange column and eluted in acontinuous linear salt gradient from 0.005 to 0.1 M pyrophosphatebuffer, pH 8.6.

The sizing of the ultrafiltration membrane and the order of use of thechromatographic columns are both important. According to the majority ofstudies, the M.W. of XO is about 300,000 daltons (Waud et al., 1975;Nagler and Vartanyan, (Biokhimiya 38:561-567, 1973 - translated fromRussian) with two subunits of about 150,000 daltons (Nathans and Hade,1975; Nagler and Vartanyan, 1976). In turn, this subunit of XO may be adimer of 80,000-85,000 daltons. This is supported by the fact that theultrafiltrate of XO preparations passed through an Amicon XM-100Amembrane (with a nominal cutoff of 100,000 daltons) contained active XO(Nathans and Hade, 1975; Biasotto and Zikakis (J. Dairy Sci. 58:1238,1975). This suggests that XO may be a tetramer which may undergodissociation and reassociation. Thus, to avoid the loss of monomers ofXO and increase the yield, the Amicon XM-50 membrane (nominal cutoff50,000 daltons) was used in this procedure. This membrane retainsmolecules with M.W. above 50,000 and allows all others to pass through.The first column used is Sephadex G-75 which retains salts and lightparticles and allows heavy particles (including XO) to come out first.Both Sepharyl S-200 and Sepharose 6B columns separate proteins in thepreparation according to molecular size. Before the final purificationstep through DEAE Sephadex A-50 (or through DEAE Sepharose CL-6B)anionic exchange column, the XO preparation is passed again throughSephadex G-75 to remove all salts (this is a necessary step; otherwise,XO will not bind to the anionic exchange column).

The final enzyme preparation has on the average a constant ratio of E₂₈₀/E₄₅₀ of 4.1, and one symmetric peak by gel chromatography. Analysis onpolyacrylamide disc gel electrophoresis shows a single band. The averageyield of the final product is 21%, which is about 110% higher than thatobtained using the best available prior art; which also produces a lesspure enzyme. Analyses on polyacrylamide disc gel electrophoresis revealthat the commercial XO migrates faster than does XO prepared by thismethod. Also, commercial XO contains 7-14 bands depending on the batch.Using neotetrazolium chloride dye, it was found only one band in thecommercially prepared XO contained activity, while the rest wereimpurities.

UTILITY OF INVENTION

Milk xanthine oxidase is of great interest because of its complexity andcatalytic versatility. The enzyme has low specificity for substrates andelectron acceptors. It catalyses the oxidation of many purines,pteridines, aldehydes, and other heterocyclic compounds by a number ofelectron acceptors such as oxygen, NADH, dyes, ferricyanides, andcytochrome C (Avis et al., J. Chem. Soc. (London) Part I: 1212-1219,1956; Corran et al., Biochem. J. 33:1694-1706, 1939; Murrey et al., J.Biol. Chem. 241:4798-4801, 1966). The presence of XO in excess or itsabsence, inhibition, or stimulation, reflects on the biochemistry ofnormal or abnormal cellular activity (Wyngaarden and Kelley, In TheMetabolic Basis of Inherited Disease, 3rd Ed., McGraw-Hill Book Co. NewYork, N.Y., 1972). Although the primary pathway of uric acid productionis known, the metabolic importance of XO is not fully understood.Recently it has been theorized that XO in bovine milk is a factor in thedevelopment of atherosclerosis in humans (Oster, Amer. J. Clin. Res.2:30-35, 1971; In Myocardiology Vol. 1, pp. 803-813, University ParkPress, Baltimore, Md., 1972; Ross et al., Proc. Soc. Exp. Biol. Med.144:523-527, 1973). The production of high purity XO will stimulateresearch in the above fields and may lead to clinical and industrialapplications. A major producer of XO has recently discontinuedproduction because of difficulty in producing acceptable XO. A processbetter than the prior art is needed.

The following example describes in greater detail the preferred processsteps used in carrying out the process of this invention.

EXAMPLE

1. To one liter of fresh raw milk (from the University of DelawareGuernsey herd) 10 ml of 200 mM sodium salicylate and 0.1 gm EDTA addedand mixed. Sodium salicylate stabilizes XO while EDTA chelates heavymetal contaminants. One liter of 0.2 M potassium phosphate buffer (pH7.8), containing 8 mM sodium salicylate and 4 mM cysteine-HCL, was addedto the mixture and mixed. The final concentration of solutes in this 2liter mixture was 5 mM sodium salicylate, 0.005% EDTA, 0.1 M K₂ HPO₄,and 2 mM cysteine-HCL. The pH of the mixture ranged between 7.8 to 7.9.

2. The mixture was incubated while stirring at 40° to 45° C. for 2hours. After 105 minutes incubation, 1% (V/V) Triton X-100 was added tothe mixture and the mixture allowed to continue incubation for 15minutes. Triton X-100 is a mild non-ionic detergent which is effectivein dissolving the MFGM. Triton X-100 is a substitute for the muchharsher lipolytic enzymes (which may adversely effect the purity of XO)and butanol (which is a denaturant and a substance difficult to workwith) presently used in other methods. At the end of the two-hourincubation, the mixture was cooled to 4° C. and, unless statedotherwise, all subsequent steps of the method were carried out at thistemperature.

3. 400 gm of solid ammonium sulfate (20% W/V) was added to the mixturewith stirring. The suspension was stirred for 15 minutes and thencentrifuged at 12,225 g for 20 minutes in an International RefrigeratedCentrifuged (Model B-20). Three distinct layers were formed aftercentrifugation. The upper layer (the milkfat) and the white precipitate(the caseins) at the bottom of the tubes were devoid of XO activity andwere discarded. The supernatant liquid was passed through glass woolinto a graduated cylinder. The filtrate was an opalescent yellow fluidwhich contained all the XO activity.

4. The concentration of ammonium sulfate in the filtered supernatantliquid was adjusted from 20% to 27% with solid ammonium sulfate, themixture stirred for 15 minutes and centrifuged at 12,225 g for 20 min.The resultant brownish-red precipitate was dissolved in 10 to 15 ml of0.1 M Tris-HCl buffer (pH 7.0) containing 2 mM sodium salicylate and0.07 M CaCl₂ and stored for at least 15 hours at -20° C. The objectiveof this step was the precipitation of caseins (Ball, 1939). About 80% ofthe total protein in cow's milk is casein which precipitates over therange of 20 to 26.4% (W/V) ammonium sulfate (McKenzie, In Milk ProteinChemistry and Molecular Biology, Vol. 2 pp. 87-114,, Acad. Press, NewYork, N.Y., 1971). This fractionation range is close to the 27% W/Vammonium sulfate used to precipitate XO in this procedure. Therefore,the inclusion of some caseins in the above precipitations isunavoidable.

5. Upon thawing the mixture to 22° C., it yielded a course whiteprecipitate of caseins. Upon centrifugation at 12,225 g and 4° C. for 20min., the mixture yielded a reddish-brown supernatant liquid and aslightly brown precipitate. The precipitate was redissolved in 0.1 MTris/CaCl₂ buffer and recentrifuged. The supernatant liquid from bothcentrifugations was combined and showed high activity of XO, while thewhite precipitate of caseins had negligible activity. It was found thatthe longer the preparation was frozen, the more caseins can be removed.Maximum casein precipitation occurs after about 3 to 4 weeks of storageat -20° C. Therefore, the time of cold storage is a function ofeconomics and the desired purity. The precipitation of casein is veryslow after 7 days and some decomposition of XO will occur, even at -20°C. Storage of most batches were from 15 hours to 1 week, which isusually a satisfactory operating range.

6. The active reddish-brown supernatant obtained in step 5 wasconcentrated to 5 ml on an Amicon ultrafiltration system using a XM50membrane designed to retain molecules of 50,000 daltons and greater.This concentrate was then applied on a Sephadex G-75 superfine column(1.5×125 cm), which has been equilibrated with 0.1 M pyrophosphatebuffer pH 7.1, and the column was eluted with the same buffer. Thepurpose of this chromatographic step was to desalt (remove the ammoniumsulfate) and remove low molecular weight (<75,000 daltons) impuritiesfrom the sample. All fractions were analyzed individually at 280 nm forprotein and at 450 nm for flavin adenine dinucleotide (FAD) on either aBeckman DB or a Gilford Model 250 spectrophotometer. From this point on,the enzyme activity in each fraction was measured spectrophotometricallyat 295 nm and 23.5° C. Fractions with activity were pooled andconcentrated by ultrafiltration as above to 5 ml. The pooled sample wasanalyzed for activity, absorption spectra, total protein, andelectrophoretic behavior.

7. The pooled, concentrated sample from step 6 was applied on aSephacryl S-200 superfine or on a Sephadex G-200 column (2.5×100 cm)equilibrated with 0.1 M pyrophosphate buffer 7.1, and the column waseluted with the same buffer. All fractions were analyzed as above.Fractions with no activity were discarded. The remaining fractionsshowed increases in purity as judged by the protein flavin ration (PFR),activity-flavin ratio (AFR), activity-protein ratio (APR), andelectrophoresis.

8. The fractions from the Sephacryl S-200 (or the Sephadex G-200) (step7) showing XO activity at 295 nm and flavin at 450 nm, were pooled,concentrated, and applied on a Sepharose 6B column (2.5×100 cm)equilibrated and eluted with 0.1 M phrophosphate buffer pH 7.1 Followinganalyses of eluted fractions, those with XO activity were pooled andconcentrated to about 3 ml by ultrafiltration using a XM50 membrane.

9. The concentrated sample of step 8 was desalted by passing it througha Sephadex G-75 column (0.9×60 cm) equilibrated and eluted with a 0.005M sodium pyrophosphate buffer pH 8.6. Fractions containing the enzymewere pooled and concentrated on a XM50 membrane.

10. The concentrated sample was applied on a DEAE Sephadex 50A (or on aDEAE Sepharose CL-6B) anionic exchange column (1.6×20 cm) which wasequilibrated with 0.005 M sodium pyrophosphate buffer pH 8.6. Initialelution of the column was with the 0.005 M phosphate buffer. At this pHand salt concentration, XO is effectively bound to the exchanger as wasapparent from the appearance of a dark brown band in the upper 2 to 4 cmof the column and its failure to elute in 0.005 M salt. Elution of XOfrom the column was accomplished on a linear continuous salt gradientfrom 0.005 M to 0.1 M sodium pyrophosphate pH 8.6. Typical data obtainedat various stages of XO preparation are listed in Table 1.

                                      Table 1                                     __________________________________________________________________________    TYPICAL DATA OBTAINED AT VARIOUS STAGES OF                                    XANTHINE OXIDASE PURIFICATION FROM GUERNSEY MILK                                         TOTAL ACTIVITY                                                                VOLUME                                                                              (1 UNITS/                                                                            PROTEIN                                                                             SPEC.                                           PROCEDURE  (ml)  ml)    (mg/ml)                                                                             ACTIVITY                                                                             PFR.sup.1                                                                         PURIFICATION                         __________________________________________________________________________    WHOLE MILK 1000  0.034* 22.66 0.0015 **  0                                    AFTER BUFFER                                                                  ADDITION   2000  0.017* 11.32 0.0015 **  0                                    AFTER                                                                         DIGESTION  2000  0.104* 12.60 0.0083 **  5.6                                  20% CUT    1862  0.115* 2.26  0.051  **  34.0                                 7% CUT     6.5   31.89  18.90 1.687  24.5                                                                              1125.0                               G-75       6.0   18.43  18.60 0.991  16.0                                                                              660.6                                SEPHACRYL S-200                                                               OR         6.0   17.71  12.40 1.428  10.0                                                                              952.0                                SEPHADEX G-200                                                                SEPHAROSE 6B                                                                             3.5   20.71  12.40 1.670  8.0 1113.3                               DEAE.sup.+ 3.5   12.20  1.70  7.176  4.1 4784.3                               __________________________________________________________________________     .sup.1 PROTEIN FLAVIN RATIO.                                                  *ACTIVITY OF SAMPLES PRIOR TO THE 7% AMMONIUM SULFATE CUT WAS DETERMINED      POLAROGRAPHICALLY.                                                            **THE PFR COULD NOT BE CALCULATED FOR SAMPLES PRIOR TO THE 7% AMMONIUM        SULFATE CUT DUE TO THE TURBIDITY OF THE SAMPLE.                          

The most sensitive indicator of XO purity is the PFR (E₂₈₀ /E₄₅₀) value(Hart et al., 1970). A decrease in the concentration of non-XO protein(at 280 nm) and a simultaneous increase in the concentration of XO (at450 nm) should give an increasingly lower E₂₈₀ /E₄₅₀ ratio. Thus, thelower the PFR value, the higher the purity of the preparation. PFRvalues obtained by various methods are: 6.2 (Corran et al., 1939), 6.2(Morell. Biochem. J. 51:657-666, 1952), 5.0 (Avis et al., 1955), 5.4(Gilbert and Bergel, 1964), 5.1 (Nelson and Handler, 1968), 5.2 (Naglerand Vartanyan, 1973), and 4.8 (Waud et al., 1975).

The enzyme purified by this method had on the average a constant ratioof E₂₈₀ /E₄₅₀ of 4.1 (the lowest PFR value ever reported), one symmetricpeak by ion-exchange chromatography, and its behavior on polyacrylamidedisc gel electrophoresis showed a single active band. Comparativeanalyses of commercially available XO, and of XO as prepared by thismethod, revealed that commercial XO migrates faster in electrophoresisthan does XO by this method. Furthermore, the commercial enzymecontained 7-14 bands depending on the batch. A rapid method was devisedto localize activity of XO directly on electrophoretic gels usingneotetrazolium chloride dye. This method involved discontinuouspolyacrylamide gel electrophoresis.

The discontinuous polyacrylamide gel electrophoresis was performed usinga Buchler 18 tube Polyanalyst according to the methods of Ornstein (Ann.N.Y. Acad. Sci. 121:321, 1964) and Davis (Ann. N.Y. Acad. Sci. 121:321and 404, 1964). Pore sizes in the gels were based on the use of 3.5%acrylamide in the sample and spacer gels and 10% acrylamide in theseparating gel. Gels were run at basic pH of 8.3 using Tris/Glycinebuffer at 2.5 to 3.0 milliamps/tube. The gel was stained for thepresence of active XO bands with neotetrazolium chloride solution [250mg of neotetrazolium chloride was dissolved in 1 liter of 10 mM xanthine(in 25 mM NaOH) and the pH adjusted to 8.3. The solution was lightbrown]. The gel containing XO was not fixed in trichloroacetic acid butwere immersed for 1 to 10 hours in the dye mixture. When the gelscontained enzymatically active XO, the process resulted in the formationof a purple color band. Detection of active XO by neotetrazoliumchloride involves the direct transfer of electrons from the substrate(xanthine) first to the flavin adenine dinucleotide moiety of the enzymeand then to neotetrazolium chloride. As a result of this electrontransfer, the purple color precipitate formazan is formed.

Gels stained as above were removed from the stain solution and immersedin distilled water for photography. The colored bands in the gelremained visible at 4° C. for up to 3 months, but faint out at roomtemperature in 2 to 6 days, depending on the activity of the enzymestained. Using this technique, commercially prepared XO was analyzedelectrophoretically and found that only one band (out of 7-14) containedXO activity, the rest were impurities.

The PFR value of the final XO preparation ranged from 2.7 to 4.8 andaveraged 4.1. The yield of the method ranges from 18-26% and average21%. Therefore, this method produces XO which is on the average about20% purer (about 4800 fold purified) and yields about 110% more XO thanthe best available method in literature (Waud et al., 1975).

More highly purified XO with PFR value approaching 2.0 can be obtainedby this method using additional treatment in columns, but the yield issmaller and the cost of the product is greater.

It is apparent that changes and modifications may be made withoutdeparting from the invention in its broader aspects. The aim of theappended claims, therefore, is to cover all such changes andmodifications as fall within the true spirit and scope of the invention.

I claim:
 1. A process for the isolation and purification of xanthineoxidase from milk which comprises first incubating the milk in thepresence of sodium salicylate, ethylenediaminetetraacetate, and aphospate buffer and then adding a non-ionic detergent to the milk andallowing it to incubate for a shorter period of time, reducing thetemperature to stop incubation and adding sufficient ammonium sulfate toprecipitate the caseins but not the xanthine oxidase, separating thecaseins and other solids and adding sufficient ammonium sulfate to thesupernatant liquid to precipitate the xanthine oxidase, separating thered-brown precipitate and dissolving same in a Tris/CaCl₂ bufferfollowed by storing the solution at freezing or below for at least 15hours to effect further separation of casein upon thawing, separatingthe supernatant liquid containing the xanthine oxidase and filteringsaid liquid with an ultrafilter which will retain molecules of 50,000daltons and greater, applying the concentrated retained material on anequilibrated chromatographic column, eluting the column with a buffersolution, collecting the eluent in multiple fractions, testing thefractions for xanthine oxidase activity, combining the samples that showacceptable activity, and continuing this process with chromatographiccolumns of varying characteristics until the desired xanthine oxidasepurity is obtained.
 2. In a process of recovering xanthine oxidase frommilk wherein milk is first incubated in the presence of sodiumsalicylate, ethylenediaminetetraacetate and phosphate buffer, theimprovement comprises adding a non-ionic detergent to the milk after thefirst stage of incubation and further allowing it to incubate for ashorter period of time before reducing its temperature.
 3. The processof claim 1 wherein the red-brown precipitate dissolved in a Tris/CaCl₂buffer is stored at -20° C. for 1 to 7 days.
 4. In a process ofrecovering xanthine oxidase from the crude mixture remaining after theremoval of most of the casein through long-term precipitation at -20° C.and centrifugation, the improvement comprises filtering with anultrafilter which will retain molecules of 50,000 daltons and greater,applying the concentrated retained material on an equilibratedchromatographic column, eluting the column with a buffer solution,collecting the eluent in multiple fractions, testing the fractions forxanthine oxidase activity, combining the samples that show acceptableactivity, and continuing this process with chromatographic columns ofvarying characteristics until the desired xanthine oxidase purity isobtained.